Rice (Oryza sativa) is one of the most important staple crops in the world, feeding almost half of the world's population, and it serves as a model for monocots, which include many important agronomic crops (e.g. wheat, maize, sorghum, millet). Food and Agriculture Organization (FAO) predicts that rice yield will have to be increased 50-70% by 2050 to meet the demands. Several approaches are currently adopted to increase rice yields, such as heterosis breeding, population improvement, wide hybridization, genetic engineering, and molecular breeding1. Among these, hybrid rice is being considered the most promising one (15-20% increases in yield)2. Crops produced from F1 hybrid seeds offer significant benefits in terms of yield improvement, agronomic performance and consistency of end-use quality. This is due to the ‘hybrid vigor’ generated by combining carefully selected parent lines. Hybrid crops are responsible for a dramatic increase in global crop yields in the past decades, and male sterility (MS) has played a significant role in this advancement. Male sterile traits can be divided into cytoplasmic male sterility (CMS), which is determined by cytoplasmic factors such as mitochondria, and genetic male sterility (GMS), which is determined by nuclear genes. CMS has long been used in hybrid corn production, while both CMS and GMS are currently used for hybrid rice production3, due to the convenience of controlling sterility expression by manipulating the gene-cytoplasm combinations in any selected genotype. Most importantly, it evades the need for emasculation in cross-pollinated species, thus encouraging cross breeding and producing pure hybrid seeds under natural conditions. However, commercial seed production must be simple and inexpensive, and the requirement for a maintainer line to produce the seed stocks of CMS line increases the production cost for this 3-line hybrid system.
On the other hand, genetic MS (GMS), controlled by nuclear genes, offers an alternative hybrid seed production system. For the two-line hybrid system, it is beneficial to use photoperiod- or temperature-inducible MS (PGMS or TGMS) mutants to maintain seed stocks for hybrid seed production. Currently, in China, PA64S is the most widely used maternal line in two-line hybrid rice breeding, and it is crossed with paternal line 93-11 to generate superhybrid rice, LYP94. PA64S, derived from a spontaneous PGMS japonica mutant NK58S (long day->13.5 h; Shi, 1985), is also a TGMS indica rice, whose MS is promoted by temperatures greater than 23.5° C., but recovers its fertility at temperatures between 21˜23° C. Recent mapping analyses demonstrate that the P/TGMS in these MS lines is regulated by a novel small RNA5. In the case of another rice genic MS mutant discovered recently, Carbon Starved Anther (CSA), the mutation on the R2R3 MYB transcription regulator defects pollen development6 and further study shows that csa is a new photoperiod-sensitive mutant, exhibiting MS under short-day conditions but male fertility under long-day conditions7. The molecular basis of its MS sensitivity to day length remains to be addressed.
Transgenic male sterility has been generated using a number of transgenes, but its application in commercial production of hybrid seeds is limited due to the lack of an efficient and economical means to maintain the MS lines, or the lack of suitable restorers8. Recently, a reversible MS system has been demonstrated in transgenic Arabidopsis plants by manipulating a R2R3 MYB domain protein (AtMYB103)8. Blocking the function of AtMYB103 using an insertion mutant or an AtMYB103EAR chimeric repressor construct under the control of the AtMYB103 promoter resulted in complete MS without seed setting8. A restorer containing the AtMYB103 gene driven by of a stronger anther-specific promoter was introduced into pollen donor plants and crossed into the MS transgenic plants for the repressor. The male fertility of F1 plants is restored. The chimeric repressor and the restorer constitute a reversible MS system for hybrid seed production. The successful application of this system for large scale hybrid seed production depends on whether the MS female parent lines can be multiplied efficiently and economically. Alternatively, an inducible promoter by chemicals or other factors (e.g. photoperiod or temperature) can be directly used to regulate the expression of a GMS gene (e.g., bHLH142) and control pollen development in transgenic plants, eliminating the costly need to maintain MS lines.
Rice anthers are composed of four lobes attached to a central core by connective and vascular tissue. When anther morphogenesis is completed, microsporocytes form in the middle, surrounded by four anther wall layers: an epidermal outer layer, endothecium, middle layer, and tapetum9. The tapetum is located in the innermost cell layer of the anther walls and plays an important role in supplying nutrients such as lipids, polysaccharides, proteins, and other nutrients for pollen development10. The tapetum undergoes programmed cell death (PCD) during the late stage of pollen development11; this PCD causes tapetal degeneration and is characterized by cellular condensation, mitochondria and cytoskeleton degeneration, nuclear condensation, and internucleosomal cleavage of chromosomal DNA. Tapetal PCD must occur at a specific stage of anther development for normal tapetum function and pollen development, and premature or delayed tapetal PCD and cellular degeneration can cause male sterility3,12-14.
Genetic and functional genomic studies of MS in Arabidopsis have shown that many transcription factors (TFs) play an essential role in pollen development and the regulation of tapetal PCD, such as mutations in DYSFUNCTIONAL TAPETUM 1 (DYT1), Defective in Tapetal Development and Function 1 (TDF1, AtMYB35), ABORTED MICROSPORES (AMS, homolog of TDR1 in rice), and MALE STERILITY 1 (MS1); and mutations in these factors all result in MS phenotype. The genetic regulatory pathway of pollen development suggests that DYT1, TDF115 and AMS16 function at early tapetum development, while MS18817 and MS115,18,19 play important roles in late tapetum development and pollen wall formation. Whilst, in rice, several TFs, such as Undeveloped Tapetum1 (UDT1, homolog of DYT1), are known to be key regulators of early tapetum development20. In addition, mutations in TAPETUM DEGERATION RETARDATION (TDR1)14, GAMYB21,22, ETERNAL TAPETUM 1 (EAT1)23 and DELAYED TAPETUM DEGENERATION (DTD)24 all cause MS associated with tapetal PCD. TDR1, ortholog of the Arabidopsis AMS gene, plays an essential role in tapetal PCD in rice; and tdr1 shows delayed tapetal degeneration and nuclear DNA fragmentation as well as abortion of microspores after release from the tetrad. Molecular evidences indicate that TDR1 directly binds the promoter of CP1 and C6 for their transcription14. C6 encodes a lipid transfer protein that plays a crucial role in the development of lipidic orbicules and pollen exine during anther development17. CP1 is involved in intercellular protein degradation in biological system and its mutant shows defected pollen development25. EAT1 acts downstream of TDR1 and directly regulates the expression of AP25 and AP37, which encode aspartic proteases involved in tapetal PCD23.
The basic helix-loop-helix (bHLH) proteins are a superfamily of TFs and one of the largest TF families in plants. There are at least 177 bHLH genes in the rice genome26,27 and more than 167 bHLH genes in Arabidopsis genome28,29. Generally, eukaryotic TFs consist of at least two discrete domains, a DNA binding domain and an activation or repression domain that operate together to modulate the rate of transcriptional initiation from the promoter of target genes30. The bHLH TFs play many different roles in plant cell and tissue development as well as plant metabolism3. The HLH domain promotes protein-protein interaction, allowing the formation of homodimeric or heterodimeric complexes31. They bind as dimers to specific DNA target sites and are important regulatory components in diverse biological processes29. So far, three of the bHLH TFs have been shown to be involved in rice pollen development—UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141).
From a screening of T-DNA tagged rice mutant pool of TNG6732, we isolated a novel MS-related gene encoding for another member of the bHLH TFs (bHLH142). In this invention, the molecular mechanism of MS in this mutant is elucidated, and it suggests that bHLH142 is specifically expressed in the anther and bHLH142 coordinates with TDR1 in regulating EAT1 promoter activity in transcription of protease genes required for PCD during pollen development. That is to say, bHLH142 plays an essential role in rice pollen development by controlling tapetal PCD. Both null mutant and overexpression transgenic plants showed a completely male sterile phenotype. Most interestingly, the overexpression plants have restored the fertility under low temperature. Homologs of SEQ ID NO: 2 with high similarity are found in other major cereal crops, and its use may increase the productivity of cereal crops by manipulating the bHLH gene for development of male sterility and production of hybrid crops.